Background: Novel approaches for prevention of prostate cancer are attractive to reduce disease-related cost, morbidity, and mortality associated with this malignancy in American men. Long-term objective of this research project is to develop a safe and effective regimen for prevention of prostate cancer using D,L- sulforaphane (SFN), which is a synthetic racemic analogue of cruciferous vegetable (e.g., broccoli) constituent L-sulforaphane. Research in the previous funding period was primarily focused on in vitro studies designed to gain insight into the mechanism by which SFN causes demise of prostate cancer cells as well as in vivo experiments to determine its efficacy for prevention of prostate cancer using a transgenic mouse model (TRAMP mice). Both these research directions were highly fruitful culminating with seminal discoveries concerning molecular anticancer pharmacology of SFN. For example, we demonstrated convincingly that the SFN-induced apoptosis in human prostate cancer cells is intimately linked to production of reactive oxygen species (ROS) due to inhibition of complex II and III of the mitochondrial respiratory chain. We also discovered that SFN treatment triggers autophagy in prostate cancer cells that serves to protect against apoptotic cell death. We are equally excited by our findings that orally administered SFN confers significant protection against prostate cancer development in TRAMP mice without any toxicity, and SFN treatment suppresses cellular pathways implicated in emergence of castration-resistant prostate cancer (CRPC). Research design in the present renewal application is largely shaped by these exciting and mostly published observations with a shift in emphasis from molecularly-oriented cellular studies to in vivo validation of the mechanistic findings with transparent translational implications. Hypothesis: Primary working hypothesis unifying the specific aims of this application predicts that SFN is empowered to not only prevent prostate cancer development by causing ROS-dependent apoptosis in cancerous cells, which is amenable to augmentation by pharmacologic suppression of autophagy, but also has the ability to inhibit emergence of CRPC by targeting both androgen receptor (AR)-dependent and AR- independent mechanisms. Specific Aims: The specific aims are to: (1) obtain in vivo evidence for contribution of ROS in proapoptotic anticancer effects of SFN using orthotopic models of human prostate cancer xenografts; (2) determine the molecular mechanism(s) of SFN-induced autophagy using relevant cellular systems; (3) determine the impact of pharmacologic suppression of autophagy on proapoptotic and chemopreventive responses to SFN using TRAMP mice; and finally (4) determine the effect of SFN administration on emergence of CRPC using castrated TRAMP mice. Translational Impact of the Proposed Research: Even though the studies conducted thus far provide compelling preclinical evidence for effectiveness of SFN against prostate cancer, efficient translation of these observations into a clinical setting is critically dependent on in vivo validation of the cellular mechanistic observations. Clinical trial design without a full appreciation of the molecular pharmacology of SFN may be sub-optimal and inherits risk of disappointment. Merit of in vivo validation of the contribution of ROS in proapoptotic anticancer effect of SFN (Aim 1) resides in regimen optimization to eliminate potential adverse drug-drug interactions between SFN and other anti-oxidants. Intrinsic value of the studies proposed in Aims 2 and 3 is defined by possible discovery of biomarkers of SFN response potentially useful in future clinical investigations as well as rational design of combination regimens involving SFN and autophagy inhibitors to achieve even greater chemopreventive efficacy. Finally, the possibility that Aim 4 ultimately leads to a novel SFN-based regimen for clinical management of CRPC is equally meritorious.